Birth–Death Dynamics of Microsatellites: Mechanistic Insights from Orthologous Loci in Felidae

<b>Background/Objectives</b>: The mutational dynamics of microsatellites over deep evolutionary timescales are poorly understood. This study aims to elucidate the life history of trinucleotide microsatellites by tracing orthologous loci across divergent vertebrate lineages and characterizing their mutational pathways. <b>Methods</b>: We developed a bioinformatic framework for identifying orthologous microsatellite loci using conserved flanking sequences. This approach was applied to three trinucleotide microsatellites located in exonic, intronic, and intergenic regions, respectively. These loci were amplified and sequenced across 126 individuals representing 64 vertebrate species, whose divergence times range from 6 to 150 million years ago (MYA). <b>Results</b>: Flanking sequences proved essential for reliable orthology assignment, while repeat motifs revealed distinct mutational pathways. Microsatellite decay occurs through two primary mechanisms: the complete loss of dominant repeats or their progressive reduction to solitary units (≤1 repeat). This degeneration process is facilitated by cryptic simple sequences (CSS), which act as genomic catalysts promoting birth-death transitions. Large intra-motif deletions were identified as the key mutational event driving contractions and eventual locus degeneration. Furthermore, mutational patterns were highly locus-specific, influenced by genomic context. <b>Conclusions</b>: Although the study focused on only three loci, limiting broader generalizations, our findings provide mechanistic insights into microsatellite evolution. These results establish a foundation for modeling complex microsatellite life histories and highlight the role of CSS in facilitating evolutionary turnover.